The complete nucleotide sequence of a conjugative and integrative plasmid, pSLS, in the thiostrepton-producing Streptomyces laurentii ATCC31255 was determined.The circular DNA molecule was 15,398 bp in length and contained 70.8"/* G+C content.Computer-assisted analyses indicated that pSLS contained 10 open reading frames (ORFs), orfl to orflO, Iocated on both strands of pSLS.The orfl encoded for a predicted protein (200 aa) showed high sirnilarity to TraA protein of non-integrative plasmid pJVI in Streptomyces phaeochromogenes involved in plasmid transfer and pock-forrnation.The orf: coded for a protein of 660'aa that shared homology with TraB proteins encoded by other Streptomyces plasmids.Proteins encoded by o,j (170 aa), orf4 (408aa) and orf5 (148aa) shared homology with Streptomyces proteins, SpdB1.SpdB2 and SpdB3, respectively, which were also involved in plasmid spreading.The orf8 encoded a polypeptide of 458 aa that shared homology wlth Int protein of integrative plasmid pSAM2 in Streptomyces ambofacience.This report manifests that pSLS is unique chimeric episomal element that contains gene cluster from non-integrative plasmid and genes for site-specific integration from integrative plasmid.
A halotolerant bacterium isolated from Thai fish sauce, obtained in Surathani province, was identified as a Pseudomonas sp. No. 3241. This strain showed halophilic ribonuclease activity. When casamino acids (CA) and yeast extract (YE) were used as the nitrogen source in a mini jar fermenter, a ratio concentration of CA to YE of 15:20 g/l in Sehgal and Gibbons Complex (SGC) medium, without NaCl, gave maximum growth and ribonuclease activity (18.18 U/ml). The ribonuclease enzyme was optimal at pH 10.0 and at the temperature of 50°C. It had marked halophilic enzyme properties that required an optimal NaCl concentration of 3 M. The ribonuclease was stable between pH 6.0 and 9.0 and at temperatures between 30 and 40°C.
The optimum condition for the protoplast formation and regeneration of S(yeptomyces Laurentii ATCC 31255 was studied.The effective formation and regeneration of protoplasts were obtained by using the mycelia grown to the stationary phase in the presence of 0.4% glycine, and by treatment with 1 mg/ml of lysozyme in PWP buffer.When plasmids, pMCP5 and its derivative pYK3 (smaller in size) having a kanamycin resistance gene, were transformed into the protoplasts by using 20% (V/V) PEG 2000 in PWP buffer, the high number of transformants were obtained at a frequency of 4.3 x lo5 and 3.4 x lo4 per pg DNA of pMCP5 and pYK3, respectively.
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